dna vaccines, methods and protocols

Purification of Supercoiled Plasmid Anthony P Green Introduction Current technologies for the purification of supercoiled plasmids are limited The use of cesium chloride gradients in the presence of ethidium bromide is time consuming, labor intensive, requires the use of known mutagens and is not conducive to large scale As a result, first-generation high-performance liquid chromatography (HPLC) methods based on anion-exchange and size exclusion have been developed but are difficult to accommodate production at large scale and still result in compromised purity (1,2) The success of DNA vaccines in animal models and the initiation of human trials (3,4) has led to a need to increase the level of supercoiled plasmid purity as well as the methodology utilized to produce these plasmids at large scale Several parameters of the purification process need to be addressed: • The ability to prepare supercoiled plasmid at purity levels acceptable for clinical material • The ability to prepare clinical grade supercoiled plasmid that will be scalable in order to produce gram quantities of product • The ability to prepare clinical grade supercoiled plasmid in accordance with cGMP principles • The ability to develop validated assays to assess purity, yield, and contamination levels Challenges to the successful development of a purification process can be divided into biological and practical The biological challenge arises from the spectrum of biomolecules that must be purified away from the supercoiled plasmid product ( Table 1) Additionally, the spectrum of nucleic acid contaminants and plasmid isoforms within that spectrum, as shown in Table 2, must be removed The removal of the relaxed DNA, DNA catenanes as well as endotoxins (5,6) are a particular problem requiring additional steps in the process From: Methods in Molecular Medicine, vol 29, DNA Vaccines: Methods and Protocols Edited by: D B Lowrie and R G Whalen Humana Press Inc., Totowa, NJ Green Table Constituents in Crude Lysate Table Plasmid DNA Forms Plasmid DNA Chromosomal DNA RNA Lipids Endotoxin Proteins Carbohydrates Monomer supercoiled Nicked Linear Dimers Catenanes The practical challenge arises because the purification process that is developed must produce highly pure product at high yield and must be reproducible, scalable and economical ( see Note 1) We describe a new purification process that has been used to generate clinical material using a proprietary non-porous polymer resin, PolyFlo ®, which uses principles of ion-pair reverse-phase chromatography to achieve separation based on size and charge density The process can be performed using either acetonitrile (ACN) or ethanol (EtOH) Simultaneous removal of contaminating endotoxins, chromosomal DNA, RNA, proteins, and plasmid isoforms during purification is a unique advantage of this resin This process meets the challenges for purity, yield, reproducibility, and scalability (1) Materials 2.1 Crude Starting Material The preparation of crude starting material from biomass is typically performed using acid/base extraction (7) This classic alkaline-lysis process provides material significantly reduced in protein, lipid and chromosomal DNA Newer protocols have been adopted to improve the initial purity even further, including the use of temperature shift during fermentation (8) or the addition of a second acetate precipitation (NH 4Ac) to reduce the RNA burden (9) We describe two purification methods: an ACN process using starting material in which minimal efforts have been made to reduce the RNA burden, and (2) an EtOH process in which the starting material has been reduced for RNA by anion-exchange chromatography and/or diafiltration The protocols described not incorporate the use of RNase ( see Note 2) 2.2 ACN Purification Materials Glass borosilicate chromatography column packed with PolyFlo resin (see Note 3) Starting material (ammonium acetate supernatant) (see Note 4) Purification of Supercoiled Plasmid 3 1.0 M TEAA (triethylamine acetate) pH 7.0 0.5 M KPO4 pH 7.0 1.0 M TBAP (tetrabutylammonium phosphate); Aldrich Chemicals (Milwaukee, WI) no.# 26, 810-0; M in H2O) 100% Acetonitrile (ACN, American Chemical Society (ACS) grade or equivalent) TES (20 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 5.0 mM NaCl) Equilibration buffer: 0.1 M TEAA pH 7.2, 6% ACN Wash buffer I: TES, 5% ACN 10 Wash buffer II: 0.1 M KPO4, 2.0 mM TBAP, 5% ACN 11 Wash buffer III: 0.1M KPO4, 2.0 mM TBAP, 15% ACN 12 Elution buffer: 0.1 M KPO4, 2.0 mM TBAP, 25% ACN 13 Sanitization buffer: 0.5 N NaOH 2.3 Ethanol Purification Materials 10 11 Glass borosilicate chromatography column packed with PolyFlo resin RNA-reduced plasmid sample 0.5 M KPO4 pH 7.0 1.0 M TBAP (tetrabutylammonium phosphate; Aldrich Chemicals # 26, 810-0; M in H2O) Ethanol (EtOH, ACS grade or equivalent) TES (0.02 M Tris-HCl pH 8.0, 1.0 mM EDTA, 5.0 mM NaCl) Equilibration buffer: 0.1 M KPO4 pH 7.0, 2.0 mM TBAP, 1% ethanol Wash buffer I: TES, 7% ethanol Wash buffer II: 0.1 M KPO4, 2.0 mM TBAP, 5% ethanol Elution buffer: 0.1 M KPO4, 2.0 mM TBAP, 25% ethanol Sanitization buffer: 0.5 N NaOH 2.4 Post-Purification Materials Millipore Pellicon II (Bedford, MA) or A/G Technology (Needham, MA) diafiltration/ultrafiltration technologies are applicable for buffer exchange and concentration Purification Protocols A schematic of the two protocols is shown in Fig 3.1 ACN Protocol Adjust to obtain a linear flow rate of 150 cm/h and equilibrate column in ≥3 column volumes of equilibration buffer Prepare sample by diluting 1/5 with TES and adjusting to 0.1 M TEAA using M TEAA stock solution Sample load should be no more than 0.5 mg/mL of resin (load concentration is based on total A260nm) Load sample and wash with equilibration buffer until the monitor returns to baseline (~2 column volumes) Collect wash Green Fig.1 Schematic flow-chart of PolyFlo purification process Wash with ~3 column volumes of wash buffer I (TES, 5% ACN) Make sure monitor returns to baseline Collect wash Wash with ~3 column volumes of wash buffer II (0.1 M KPO4, 2.0 mM TBAP, 5% ACN) Wash with ~3 column volumes of wash buffer III (0.1 M KPO4, 2.0 mM TBAP, 15% ACN) until the monitor returns to baseline Collect wash Elute product with a 10-column volumes linear gradient from 0.1 M KPO4, 2.0 mM TBAP, 15% ACN to 0.1 M KPO4, 2.0 mM TBAP, 25% ACN Collect elution fractions This is the purified product Clean column by running column volumes of sanitization buffer (0.5 N NaOH) Turn pump off and let sit in 0.5 N NaOH for h Re-equilibrate column with ≥3 column volumes of equilibration buffer Monitor pH to assure that all the NaOH has been removed Purified sample from step may be processed through concentration and/or buffer exchange steps It is recommended to diafilter against 0.5 M Na-acetate pH 7.8 to remove residual TBAP 3.2 ACN Protocol Results The results of the ACN protocol are shown in Fig No difference in the purity of the product is seen using starting material representing g or 100 g of biomass The RNA is eliminated Despite the significant quantities of relaxed DNA, >50% of total plasmid, this contaminant is removed in the wash step The final purity is >90% 3.3 Ethanol Protocol Adjust pump speed to obtain a linear flow rate of 150 cm/hr and equilibrate column in ≥ column volumes of equilibration buffer Purification of Supercoiled Plasmid Fig PolyFlo chromatography of plasmid using ACN process (A) Chromatographic tracing for application of nucleic acid sample extracted from 1.0 g E coli cells (B,C) 1% Agarose gel analysis of resolved peaks from 1.0 g biomass (B) and 100 g biomass (C) Lane = µg nucleic acid sample; lanes and = TES and ACN wash; lanes and = 15% (v/v) ACN/TBAP wash; lanes and = 15-25% (v/v) ACN gradient; and lane 10 = 50% ACN strip Reprinted from (1) Prepare sample for loading by adjusting to mM TBAP and 1% ethanol Sample load should be no more than 0.5 mg/mL of resin (load concentration is based on total A260nm) Sample should be 60% of plasmid, this contaminant is removed in the wash step The final purity is >95% Notes 4.1 Organic Solvent The choice of organic solvent for chromatography is predicated on the amount of contaminating RNA In general, if the RNA burden is less than 50%, the ethanol process may be employed This can be accomplished through anionexchange chromatography, diafiltration or RNase treatment A rigorous test of the amount of contaminating RNA below which the ethanol process can be used has not been performed If RNA reduction is achieved by RNase digestion, the sample must be diafiltered or dialyzed to remove excess ribonucleotides prior to PolyFlo chromatography Purification of Supercoiled Plasmid Fig Endotoxin binding to a PolyFlo column (1 × cm) (A) Total endotoxin units (EU) were determined in the flow-through (solid squares) and gradient elution (open circles) after loading sample buffer was spiked with increasing levels of endotoxin (B) Analysis of endotoxin levels in purified plasmid preparations at defined intervals during 100 consecutive applications of a single PolyFlo chromatography column Reprinted from (1) 4.2 Endotoxin Removal PolyFlo has an extremely hydrophobic surface As such, significant quantities of endotoxin are removed as part of the purification process and in a reproducible manner ( Fig 4) 4.3 Specifications No specifications for the purity of supercoiled plasmid, the levels of residual contaminants or even the methods for evaluating purity have been codified (10) While many methods can be used to analyze plasmid DNA, it is only recently that these methods have been applied to plasmid DNA as a potential pharmaceutical product (11) Table describes some of the target specifications and methods used within the industry 4.4 Multiple Chromatography Runs PolyFlo is a chemically inert polymer that withstands rigorous sanitization procedures which allows for multiple runs One hundred consecutive applications of crude plasmid with no change in purity or contamination levels have Green Table Target Specifications Parameter Purity % Monomer supercoiled Purity Contaminants RNA Genomic DNA Endotoxin Protein Target specification Testing method >95 1.8–2.0 1% Agarose gel A260:A280